1. Field of the Invention
The invention relates to a process for the detection of complexes of cathepsin G and .alpha.-1-antichymotrypsin in body fluids. Furthermore, the invention relates to a diagnostic kit to accomplish this process.
2. Background Art
.alpha.-1-antichymotrypsin is a 68-kDa glycoprotein and belongs to the "serpin" family (serine proteinase inhibitors) of proteinase inhibitors in plasma (Journal of Medicine 16: 101-128 (1985); Annual Review of Biochemistry 52: 655-709 (1983)). It is mainly synthesized in the liver, but can be produced by various tissues. The concentration of .alpha.-1-antichymotrypsin which is an acute-phase reactant in plasma can drastically increase within hours after injury. It inhibits serine proteinases of chymotrypsin-like specificity. Cathepsin G from neutrophiles is a primary enzyme targeted by .alpha.-1-antichymotrypsin. The association rate of .alpha.-1-antichymotrypsin for chymotrypsin is too slow to be of biological importance (Journal of Biological Chemistry 255: 3931-3934 (1980)).
Although the interrelationship of .alpha.-1-antichymotrypsin and cathepsin G is well established, the physiological role of the system is not known. The several proposed functions of cathepsin G are connective tissue turnover (Biochemical Journal 167: 629-237 (1977)) microbe elimination (Infection and Immunity 14: 1276-1283 (1976)), angiotensin-II generation (Journal of Biological Chemistry 257: 8619-8622 (1982)), turnover of clotting (Thrombocyte Research 6: 315-326 (1975)) and complement factors.
Detection of deficiencies of some other serine proteinase inhibitors homologous to .alpha.-1-antichymotrypsin has helped to explain their biological function. However, immunological measurements of .alpha.-1-antichymotrypsin fail to detect total .alpha.-1-antichymotrypsin deficiency in patients despite intensive screening. Patients with .alpha.-1-antichymotrypsin concentrations.ltoreq.50% of normal--either heterozygotes for an autosomal dominant trait or acquired by liver impairment--appear to be predisposed to lung and liver disease but no clear cause--effect conclusions can be drawn thus far. Functional impairment of .alpha.-1-antichymotrypsin could not be assessed in complex biological fluids such as plasma, because no proteinases are known which react exclusively with .alpha.-1-antichymotrypsin. This was, however, achieved by an immunoassay by which active .alpha.-1-antichymotrypsin could be specifically measured in plasma (Clinical Chemistry 36: 2077-2081 (1990)).
However, for several applications the measurement of complexes of cathepsin G and .alpha.-1-antichymotrypsin in nanomolar concentrations would be desirable in order to recognize disease states with an increased turnover of cathepsin G or .alpha.-1-antichymotrypsin. While a sensitive sandwich ELISA (Enzyme Linked Immunosorbent Assay) has been described for the analogous system of human leukocyte elastase and its primary plasma inhibitor, .alpha.-1-proteinase inhibitor (J. Clin. Chem. Clin. Biochem. 22: 693-697 (1984); EP-0 038 935), no such assay is available for complexes of cathepsin G and .alpha.-1-antichymotrypsin because of the strong avidity of cathepsin G to solid surfaces which prevented the application of cathepsin in such a classic sandwich ELISA.
The task of this invention is therefore to provide an assay for the specific identification of complexes of cathepsin G and .alpha.-1-antichymotrypsin in body fluids.